The host immune system provides a sophisticated defence mechanism that enables the recognition and elimination of foreign entities, such as infections agents or neoplasms, from the body. When functioning properly, an effective immune system distinguishes between foreign invaders and the host's own tissues. The first response to foreign agents is the secretion of antibodies that are able to recognize, block and destroy microbial agents. However, this response is often not sufficient because in some cases, such as viral particles, the pathogens are able to escape B cell antibody response by rapidly entering into target cells where the antibodies cannot reach them. The pathogen can then replicate intracellularly and infect other peripheral cells. The challenge for scientists is to enhance the T-cell response against microbial agents. T-cell response is the capacity of the immune system to raise a special type of lymphocytes [CD4+, CD8+] that are able to recognize specifically the infected cells and destroy them. This mechanism of T-cell response is complementary to the B-cell antibody response and both are needed to elicit an efficient immune response. As an example, an efficient vaccine against HIV infection is a long process because of the difficulty to generate a CTL response against various vaccine candidates.
Dendritic cells (DCs) are efficient antigen presenting cells (APC) that initiate immune response to peptide antigens associated with class I and II MHC (Freudenthal, P. S. and Steinman, R. M., Proc. Natl. Acad. Sci. USA 87:7698, 1990; Steinman, R. M., Ann. Rev. Immune. 9:271, 1991). DCs represent a small subpopulation of widely distributed, bone marrow-derived leucocytes, which are the only natural antigen presenting cells able to prime naive T cells. They activate both CD4+ and CD8+ T lymphocyte primary immune response, and are at least as effective as other APCs such as monocytes in stimulating secondary immune responses (Peters et al., Immunol. Today L7:273, 1997).
In order to stimulate T lymphocyte responses, peptide fragments from antigens contained in a vaccine must first be bound to peptide binding receptors (major histocompatibility complex [MHC] class I and II molecules) that display the antigenic peptides on the surface of antigen presenting cells (APCs). T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides. Current models of antigen processing and presentation to T lymphocytes suggest that two principle pathways exist. In brief, exogenous antigens are internalised into the endocytic compartments of APCs where they are hydrolysed into peptides, some of which become bound to MHC class II molecules. The mature MHC class II/peptide complexes are then transported to the cell surface for presentation to class II-restricted CD4+ T lymphocytes. In contrast, for the MHC class I molecules, endogenous antigens are degraded in the cytoplasm by the action of a proteolytically active particle known as the proteasome before their transport into the endoplasmic reticulum, where they bind to nascent MHC class I molecules. Stable class I/peptide complexes are transported through Golgi apparatus to the cell surface to CD8+ CTL. Because the CTL response is crucial for protection against many viral or parasitic infections and some tumour cells, several new vaccine strategies have been proposed: 1) Immunostimulating complexes (Takahasci et al. 1990. Nature 344:873); 2) antigen-loaded pH-sensitive liposomes (Nair et al. 1992. J. Exp. Med. 175:609); 3) recombinant bacteria expressing foreign antigens (Tuner et al. 1993, Infect. Immun. 61:5374; Ikonomidis et al. 1994. J. Exp. Med 180:2209); 4) bacterial toxins fused to CTL epitopes (Donnelly et al. 1993. Proc; Natl. Acad. Sci USA 90:3530); 5) particulate antigens (Schirmbeck et al. 1994. Eur. J. Immunol 24:2068, Layton et al, 1993. J. Immunol 151:1097); 6) use of various vectors (Schutze-Redelmeier et al. 1996, J. Immunology 157:650-655; Schluesener 1996, J Neurosci Res 46:258-262); and 7) naked DNA injected in muscle cells (Ulmer et al. 1993. Science 259:1745). This variety of strategies reflects the inherent difficulty of delivering antigens intracellularly in order to elicit a CTL response. In many cases, these approaches have a poor in vivo efficiency and are limited by safety considerations, immune responses against the vector, and cost.
In addition to the immune system, mammals are known to produce small peptides which have direct antimicrobial activity. Most of these peptides act by causing direct lysis of the membrane of prokaryotes. A major family of these peptides are β-stranded antibiotic peptides linked by disulphide bonds. Members of the family include defensins (Lehrer et al, 1991, Cell 64:229-230; Lehrer et al, 1993, Ann. Rev. Immunol. 11:105-128), protegrins (Kokryakov et al, 1993, FEBS 337:231-236) and tachyplesins (Nakamura et al, 1988, J. Biol. Chem. 236:16709-16713; Miyata et al, 1989, J. Biochem. 106:663-668).
Peptides of these classes are known to be able to pass through the membranes of mammalian cells, though due to the differences between bacterial and mammalian cell membranes, the peptides are non-toxic to mammalian cells.
WO99/07728 describes a number of derivatives of these peptides as vectors for the introduction of substances to cells or for substances to pass through the blood-brain barrier. These derivatives include linear derivatives in which the peptides do not have disulphide bonds. The absence of disulphide bonds is brought about by substitution of cysteine residues, or blocking their terminal thiol groups.